Methods and pharmaceutical compositions for the treatment of beta-thalassemias

ABSTRACT

The present invention relates to methods and pharmaceutical compositions for the treatment of beta-thalassemias. In particular, the present invention relates to an XPO1 inhibitor for use in a method for treating beta-thalassemia in a subject in need thereof.

FIELD OF THE INVENTION

The present invention relates to methods and pharmaceutical compositionsfor the treatment of beta-thalassemias.

BACKGROUND OF THE INVENTION

Adult mammalian Hg is a multimeric protein that includes two α and two βglobin chains which together form the (α/β)₂ tetrameric hemoglobin (Hb)molecule. Beta-thalassemias are a group of inherited blood disorderscaused by a quantitative defect in the synthesis of the β chains ofhemoglobin. In individuals with this disorder, the synthesis of β-globinchains is reduced or absent. Three main forms of the disease have beendescribed: β-thalassemia major (β-TM or β⁰-TM) in which no β chain isproduced, and β-thalassemia intermedia and β-thalassemia minor, in whichβ chain is produced but in lower than normal amounts. These conditionscause variable phenotypes ranging from severe anemia to clinicallyasymptomatic individuals. Individuals with β-TM usually present withinthe first two years of life with severe anemia, poor growth, andskeletal abnormalities during infancy. Affected children will requireregular lifelong blood transfusions. β-thalassemia intermedia is lesssevere than β-thalassemia major and may require episodic bloodtransfusions. Transfusion-dependent patients will develop iron overloadand require chelation therapy to remove the excess iron.

Despite extensive knowledge of the molecular defects causingβ-thalassemia major (TM), less is known about the mechanisms responsibleof the ineffective erythropoiesis. This latter is characterized byaccelerated erythroid differentiation, apoptosis and maturation arrestat the polychromatophilic stage. It explains, at least in part, theprofound anemia observed in this disease. Although it has been proposedthat both the precipitation of unmatched globin chains as well as theaccumulation of unbound iron could lead to oxidative stress andsubsequent hemolysis, the mechanism by which apoptosis and maturationarrest are induced remains unclear.

The present inventors have elucidated the consequences of α chaincytoplasmic accumulation and the cascade of failed reactions that resulttherefrom which ultimately cause β-TM symptoms such as anemia. Thediscovery is based on the further clarification of the roles of thechaperone protein HSP70 and the erythrocyte maturation protein GATA-1(https://ash.confex.com/ash/2012/webprogram/Paper48181.html). Theinventors have discovered that HSP70 has important functions in both thecytoplasm and the nucleus of erythroblasts. A primary function of HSP70in the nucleus is binding to the GATA-1 protein and preventing itscleavage and proteolytic degradation (by the protease caspase-3). HSP70thus prevents inactivation of GATA-1 and preserves its function as a keyfactor in erythrocyte maturation. A secondary function of HSP70 isbinding to α globin in the cytoplasm and ensuring that the proteinchains are properly folded and can form tetrameric (α/β)₂ Hb.Ordinarily, there is sufficient HSP70 available in the cell to carry outboth of these functions. However, in β-TM cells, the HSP70 ismonopolized by the excess of free α chains which accumulate in thecytoplasm. Thus, a disproportionate amount of the HSP70 is sequesteredin the cytoplasm, and there is not sufficient HSP70 available forbinding and protecting GATA-1 in the nucleus. Unprotected GATA-1 isproteolytically cleaved and inactivated, and proper erythrocytematuration does not occur. Rather, the absence of active GATA-1 resultsin maturation arrest and apoptosis of immature erythrocytes at thepolychromatophilic stage. This sequence of events is thus initiallytriggered by a lack of hemoglobin β chains and ultimately results in low(or no) erythrocyte production, causing anemia.

SUMMARY OF THE INVENTION

The present invention relates to methods and pharmaceutical compositionsfor the treatment of beta-thalassemias. In particular, the presentinvention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and pharmaceutical compositionsdesigned to intervene in this defective process and to promote orrestore erythrocyte maturation in individuals suffering from β-TM. It isnoted that because β globin is not formed in β-TM erythrocytes, the typeof erythrocytes that are produced in individuals treated with themethods and compositions described herein contain (α/γ)₂ Hb, and theinvention provides methods and compositions for increasing theproduction of (α/γ)₂ Hb erythrocytes in β-TM cells and β-TM individuals.The methods involve maintaining the activity of GATA-1 by preventingnuclear export of HSP70 in the cytoplasm. Accordingly, it is an objectof this invention to provide methods of restoring or increasingerythrocyte maturation in a subject suffering from β-thalassemia major(β-TM) by preventing proteolytic inactivation of GATA-1. In someembodiments, preventing is achieved by administering to the subject acompound that inhibits the XPO1 nuclear transporter.

The present invention relates to an XPO1 inhibitor for use in a methodfor treating beta-thalassemia in a subject in need thereof.

The present invention relates to an XPO1 inhibitor for use in a methodfor promoting or restoring erythrocyte maturation in a subject sufferingfrom beta-thalassemia.

The present invention relates to an XPO1 inhibitor for use in a methodfor increasing the production of (α/γ)₂ Hb erythrocytes in a subjectsuffering from beta-thalassemia.

As used herein the term “XPO1” has its general meaning in the art andrefers to the exportin 1 protein. The protein mediates leucine-richnuclear export signal (NES)-dependent protein transport. The protein isalso called Chromosomal Region Maintenance 1 (Crm1).

As used herein the term “XPO1 inhibitor” designates any compound ortreatment that reduces or blocks the activity of XPO1. The term alsoincludes inhibitors of XPO1 expression.

XPO1 inhibitors are well known in the art. In certain embodiments of theinvention the XPO1 inhibitor is selected from small molecule compoundsthat have been disclosed in the following publications WO2011109799,WO2012099807, WO2013020024, WO 2013019548, and WO2013019561.

In some embodiments, the XPO1 inhibitor is selected from the groupconsisting of the following compounds:

and pharmaceutically acceptable salts thereof.

In some embodiments, the XPO1 inhibitor is selected from the groupconsisting of (Z)-3-[3-(3-Chloro-phenyl)-[1,2,4]-triazol-1-yl]-acrylicacid ethyl ester;(E)-3-[3-(3-Chloro-phenyl)-[1,2,4]-triazol-1-yl]-acrylic acid ethylester; (Z)-3-[3-(3-Chloro-phenyl)-[1,2,4]-triazol-1-yl]-acrylic acidisopropyl ester;(E)-3-[3-(3-Chloro-phenyl)-[1,2,4]-triazol-1-yl]-acrylic acid isopropylester; (Z)-3-[3-(3-Chloro-phenyl)-[1,2,4]-triazol-1-yl]-acrylic acidtert-butyl ester;(Z)-3-[3-(3-Chloro-phenyl)-[1,2,4]-triazol-1-yl]-acrylic acid tert-butylester; (E)-3-[3-(3-Chloro-phenyl)-[1,2,4]-triazol-1-yl]-N-phenyl-acrylamide;(E)-N-(2-Chloro-phenyl)-3-[3-(3-chloro-phenyl)-[1,2,4]-triazol-1-yl]-acrylamide;(4-{(E)-3-[3-(3-Chloro-phenyl)-[1,2,4]-triazol-1-yl]-acryloylamino}-phenyl)-carbamicacid tert-butyl ester;(E)-3-[3-(3-Chloro-phenyl)-[1,2,4]-triazol-1-yl]-N-(4-methoxy-phenyl)-acrylamide;(E)-3-[3-(3-Chloro-phenyl)-[1,2,4]-triazol-1-yl]-N-methyl-N-phenyl-acrylamide;(E)-3-[3-(3-Chloro-phenyl)-[1,2,4]-triazol-1-yl]-N-methyl-N-phenyl-acrylamide;(E)-N-(4-Amino-phenyl)-3-[3-(3-chloro-phenyl)-[1,2,4]-triazol-1-yl]-acrylamide and/or a pharmaceutically-acceptable salt thereof.

In some embodiments, the XPO1 inhibitor is selected from the groupconsisting of the following compounds:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the XPO1 inhibitor is selected from the groupconsisting of a compound selected from the group consisting of1-(4-methoxyphenyl)-1H-pyrrole-2,5-dione;1-(4-bromo-2,5-difluorophenyl)-1H-pyrrole-2,5-dione;3-methyl-1-(1-methyl-1H-pyrazol-3-yl)-1H-pyrrole-2,5-dione;4-(2,5-dioxo-dihydro-1H-pyrrol-1-yl)-N-(5-methylisoxazol-3-yl)benzenesulfonamide;1-(3-benzoyl-4-methylthiophen-2-yl)-1H-pyrrole-2,5-dione;1-(4-(3-(trifluoromethyl)-1H-pyrazol-1-yl)phenyl)-1H-pyrrole-2,5-dione;1-(4-(4-chlorophenyl)thiazol-2-yl)-3-methyl-1H-pyrrole-2,5-dione;1-(benzo[b]thiophen-3-ylmethyl)-1H-pyrrole-2,5-dione;1-(3,4-dimethoxyphenethyl)-1H-pyrrole-2,5-dione;1-(naphthalen-1-yl)-1H-pyrrole-2,5-dione;1-(4-cyclohexylphenyl)-1H-pyrrole-2,5-dione;1-(2-benzoylphenyl)-1H-pyrrole-2,5-dione;1-(4-morpholinophenyl)-1H-pyrrole-2,5-dione;1-(4-chlorophenethyl)-1H-pyrrole-2,5-dione;1-(2-(thiophen-2-yl)ethyl)-1H-pyrrole-2,5-dione;1-([3,4]methylenedioxybenzyl)-1H-pyrrole-2,5-dione amide and/or apharmaceutically-acceptable salt thereof.

In some embodiments, the XPO1 inhibitor is selected from the groupconsisting of the following compounds:

In some embodiments, the XPO1 inhibitor is KPT-330 (Etchin J, Sanda T,Mansour M R, Kentsis A, Montero J, Le B T, Christie A L, McCauley D,Rodig S J, Kauffman M, Shacham S, Stone R, Letai A, Kung A L, ThomasLook A. KPT-330 inhibitor of CRM1 (XPO1)-mediated nuclear export hasselective anti-leukaemic activity in preclinical models of T-cell acutelymphoblastic leukaemia and acute myeloid leukaemia. Br J Haematol. 2013April; 161(1):117-27. doi: 10.1111/bjh.12231. Epub 2013 Feb. 4).

In some embodiments, the XPO1 inhibitor is KPT-276 (Schmidt J, BraggioE, Kortuem K M, Egan J B, Zhu Y X, Xin C S, Tiedemann R E, Palmer S E,Garbitt V M, McCauley D, Kauffman M, Shacham S, Chesi M, Bergsagel P L,Stewart A K. Genome-wide studies in multiple myeloma identify XPO1/CRM1as a critical target validated using the selective nuclear exportinhibitor KPT-276. Leukemia. 2013 Jun. 11. doi: 10.1038/leu.2013.172.).

An “inhibitor of expression” refers to a natural or synthetic compoundthat has a biological effect to inhibit the expression of a gene.

In a preferred embodiment of the invention, said inhibitor of geneexpression is a siRNA, an antisense oligonucleotide or a ribozyme.

Inhibitors of gene expression for use in the present invention may bebased on antisense oligonucleotide constructs. Anti-senseoligonucleotides, including anti-sense RNA molecules and anti-sense DNAmolecules, would act to directly block the translation of the targetedmRNA by binding thereto and thus preventing protein translation orincreasing mRNA degradation, thus decreasing the level of the targetedprotein (i.e. XPO1), and thus activity, in a cell. For example,antisense oligonucleotides of at least about 15 bases and complementaryto unique regions of the mRNA transcript sequence encoding the targetprotein can be synthesized, e.g., by conventional phosphodiestertechniques and administered by e.g., intravenous injection or infusion.Methods for using antisense techniques for specifically inhibiting geneexpression of genes whose sequence is known are well known in the art(e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323;6,107,091; 6,046,321; and 5,981,732).

Small inhibitory RNAs (siRNAs) can also function as inhibitors of geneexpression for use in the present invention. Gene expression can bereduced by contacting the tumor, subject or cell with a small doublestranded RNA (dsRNA), or a vector or construct causing the production ofa small double stranded RNA, such that gene expression is specificallyinhibited (i.e. RNA interference or RNAi). Methods for selecting anappropriate dsRNA or dsRNA-encoding vector are well known in the art forgenes whose sequence is known (e.g. see Tuschi, T. et al. (1999);Elbashir, S. M. et al. (2001); Hannon, G J. (2002); McManus, M T. et al.(2002); Brummelkamp, T R. et al. (2002); U.S. Pat. Nos. 6,573,099 and6,506,559; and International Patent Publication Nos. WO 01/36646, WO99/32619, and WO 01/68836).

Ribozymes can also function as inhibitors of gene expression for use inthe present invention. Ribozymes are enzymatic RNA molecules capable ofcatalyzing the specific cleavage of RNA. The mechanism of ribozymeaction involves sequence specific hybridization of the ribozyme moleculeto complementary target RNA, followed by endonucleolytic cleavage.Engineered hairpin or hammerhead motif ribozyme molecules thatspecifically and efficiently catalyze endonucleolytic cleavage of thetargeted mRNA sequences are thereby useful within the scope of thepresent invention. Specific ribozyme cleavage sites within any potentialRNA target are initially identified by scanning the target molecule forribozyme cleavage sites, which typically include the followingsequences, GUA, GUU, and GUC. Once identified, short RNA sequences ofbetween about 15 and 20 ribonucleotides corresponding to the region ofthe target gene containing the cleavage site can be evaluated forpredicted structural features, such as secondary structure, that canrender the oligonucleotide sequence unsuitable. The suitability ofcandidate targets can also be evaluated by testing their accessibilityto hybridization with complementary oligonucleotides, using, e.g.,ribonuclease protection assays.

Both antisense oligonucleotides and ribozymes useful as inhibitors ofgene expression can be prepared by known methods. These includetechniques for chemical synthesis such as, e.g., by solid phasephosphoramidite chemical synthesis. Alternatively, anti-sense RNAmolecules can be generated by in vitro or in vivo transcription of DNAsequences encoding the RNA molecule. Such DNA sequences can beincorporated into a wide variety of vectors that incorporate suitableRNA polymerase promoters such as the T7 or SP6 polymerase promoters.Various modifications to the oligonucleotides of the invention can beintroduced as a means of increasing intracellular stability andhalf-life. Possible modifications include but are not limited to theaddition of flanking sequences of ribonucleotides ordeoxyribonucleotides to the 5′ and/or 3′ ends of the molecule, or theuse of phosphorothioate or 2′-O-methyl rather than phosphodiesteraselinkages within the oligonucleotide backbone.

Antisense oligonucleotides siRNAs and ribozymes of the invention may bedelivered in vivo alone or in association with a vector. In its broadestsense, a “vector” is any vehicle capable of facilitating the transfer ofthe antisense oligonucleotide siRNA or ribozyme nucleic acid to thecells. Preferably, the vector transports the nucleic acid to cells withreduced degradation relative to the extent of degradation that wouldresult in the absence of the vector. In general, the vectors useful inthe invention include, but are not limited to, plasmids, phagemids,viruses, other vehicles derived from viral or bacterial sources thathave been manipulated by the insertion or incorporation of the antisenseoligonucleotide siRNA or ribozyme nucleic acid sequences. Viral vectorsare a preferred type of vector and include, but are not limited tonucleic acid sequences from the following viruses: retrovirus, such asmoloney murine leukemia virus, harvey murine sarcoma virus, murinemammary tumor virus, and rouse sarcoma virus; adenovirus,adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barrviruses; papilloma viruses; herpes virus; vaccinia virus; polio virus;and RNA virus such as a retrovirus. One can readily employ other vectorsnot named but known to the art.

Preferred viral vectors are based on non-cytopathic eukaryotic virusesin which non-essential genes have been replaced with the gene ofinterest. Non-cytopathic viruses include retroviruses (e.g.,lentivirus), the life cycle of which involves reverse transcription ofgenomic viral RNA into DNA with subsequent proviral integration intohost cellular DNA. Retroviruses have been approved for human genetherapy trials. Most useful are those retroviruses that arereplication-deficient (i.e., capable of directing synthesis of thedesired proteins, but incapable of manufacturing an infectiousparticle). Such genetically altered retroviral expression vectors havegeneral utility for the high-efficiency transduction of genes in vivo.Standard protocols for producing replication-deficient retroviruses(including the steps of incorporation of exogenous genetic material intoa plasmid, transfection of a packaging cell lined with plasmid,production of recombinant retroviruses by the packaging cell line,collection of viral particles from tissue culture media, and infectionof the target cells with viral particles) are provided in KRIEGLER (ALaboratory Manual,” W.H. Freeman C.O., New York, 1990) and in MURRY(“Methods in Molecular Biology,” vol. 7, Humana Press, Inc., Cliffton,N.J., 1991).

Preferred viruses for certain applications are the adeno-viruses andadeno-associated viruses, which are double-stranded DNA viruses thathave already been approved for human use in gene therapy. Theadeno-associated virus can be engineered to be replication deficient andis capable of infecting a wide range of cell types and species. Itfurther has advantages such as, heat and lipid solvent stability; hightransduction frequencies in cells of diverse lineages, includinghematopoietic cells; and lack of superinfection inhibition thus allowingmultiple series of transductions. Reportedly, the adeno-associated viruscan integrate into human cellular DNA in a site-specific manner, therebyminimizing the possibility of insertional mutagenesis and variability ofinserted gene expression characteristic of retroviral infection. Inaddition, wild-type adeno-associated virus infections have been followedin tissue culture for greater than 100 passages in the absence ofselective pressure, implying that the adeno-associated virus genomicintegration is a relatively stable event. The adeno-associated virus canalso function in an extrachromosomal fashion.

Other vectors include plasmid vectors. Plasmid vectors have beenextensively described in the art and are well known to those of skill inthe art. See e.g., SANBROOK et al., “Molecular Cloning: A LaboratoryManual,” Second Edition, Cold Spring Harbor Laboratory Press, 1989. Inthe last few years, plasmid vectors have been used as DNA vaccines fordelivering antigen-encoding genes to cells in vivo. They areparticularly advantageous for this because they do not have the samesafety concerns as with many of the viral vectors. These plasmids,however, having a promoter compatible with the host cell, can express apeptide from a gene operatively encoded within the plasmid. Somecommonly used plasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40, andpBlueScript. Other plasmids are well known to those of ordinary skill inthe art. Additionally, plasmids may be custom designed using restrictionenzymes and ligation reactions to remove and add specific fragments ofDNA. Plasmids may be delivered by a variety of parenteral, mucosal andtopical routes. For example, the DNA plasmid can be injected byintramuscular, intradermal, subcutaneous, or other routes. It may alsobe administered by intranasal sprays or drops, rectal suppository andorally. It may also be administered into the epidermis or a mucosalsurface using a gene-gun. The plasmids may be given in an aqueoussolution, dried onto gold particles or in association with another DNAdelivery system including but not limited to liposomes, dendrimers,cochleate and microencapsulation.

Typically the XPO1 inhibitor is administered to the subject in atherapeutically effective amount.

By a “therapeutically effective amount” of the XPO1 inhibitor of theinvention as above described is meant a sufficient amount of thecompound. It will be understood, however, that the total daily usage ofthe compounds and compositions of the present invention will be decidedby the attending physician within the scope of sound medical judgment.The specific therapeutically effective dose level for any particularsubject will depend upon a variety of factors including the disorderbeing treated and the severity of the disorder; activity of the specificcompound employed; the specific composition employed, the age, bodyweight, general health, sex and diet of the subject; the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific polypeptide employed; andlike factors well known in the medical arts. For example, it is wellwithin the skill of the art to start doses of the compound at levelslower than those required to achieve the desired therapeutic effect andto gradually increase the dosage until the desired effect is achieved.However, the daily dosage of the products may be varied over a widerange from 0.01 to 1,000 mg per adult per day. Preferably, thecompositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0,25.0, 50.0, 100, 250 and 500 mg of the active ingredient for thesymptomatic adjustment of the dosage to the subject to be treated. Amedicament typically contains from about 0.01 mg to about 500 mg of theactive ingredient, preferably from 1 mg to about 100 mg of the activeingredient. An effective amount of the drug is ordinarily supplied at adosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day,especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

The XPO1 inhibitor of the invention may be combined withpharmaceutically acceptable excipients, and optionally sustained-releasematrices, such as biodegradable polymers, to form therapeuticcompositions.

“Pharmaceutically” or “pharmaceutically acceptable” refers to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to a mammal, especially ahuman, as appropriate. A pharmaceutically acceptable carrier orexcipient refers to a non-toxic solid, semi-solid or liquid filler,diluent, encapsulating material or formulation auxiliary of any type.

In the pharmaceutical compositions of the present invention for oral,sublingual, subcutaneous, intramuscular, intravenous, transdermal, localor rectal administration, the active principle, alone or in combinationwith another active principle, can be administered in a unitadministration form, as a mixture with conventional pharmaceuticalsupports, to animals and human beings. Suitable unit administrationforms comprise oral-route forms such as tablets, gel capsules, powders,granules and oral suspensions or solutions, sublingual and buccaladministration forms, aerosols, implants, subcutaneous, transdermal,topical, intraperitoneal, intramuscular, intravenous, subdermal,transdermal, intrathecal and intranasal administration forms and rectaladministration forms. Galenic adaptations may be done for specificdelivery in the small intestine or colon.

Preferably, the pharmaceutical compositions contain vehicles which arepharmaceutically acceptable for a formulation capable of being injected.These may be in particular isotonic, sterile, saline solutions(monosodium or disodium phosphate, sodium, potassium, calcium ormagnesium chloride and the like or mixtures of such salts), or dry,especially freeze-dried compositions which upon addition, depending onthe case, of sterilized water or physiological saline, permit theconstitution of injectable solutions.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi.

Solutions comprising XPO1 inhibitors of the invention as free base orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The XPO1 inhibitor of the invention can be formulated into a compositionin a neutral or salt form. Pharmaceutically acceptable salts include theacid addition salts (formed with the free amino groups of the protein)and which are formed with inorganic acids such as, for example,hydrochloric or phosphoric acids, or such organic acids as acetic,oxalic, tartaric, mandelic, and the like. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium, or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, histidine,procaine and the like.

The carrier can also be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetables oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifusoluble agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminiummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activepolypeptides in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms, such as the type of injectable solutions described above,but drug release capsules and the like can also be employed.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media which can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion. Some variation in dosage will necessarilyoccur depending on the condition of the subject being treated. Theperson responsible for administration will, in any event, determine theappropriate dose for the individual subject.

The XPO1 inhibitor of the invention may be formulated within atherapeutic mixture to comprise about 0.0001 to 1.0 milligrams, or about0.001 to 0.1 milligrams, or about 0.1 to 1.0 or even about 10 milligramsper dose or so. Multiple doses can also be administered.

In addition to the XPO1 inhibitors of the invention formulated forparenteral administration, such as intravenous or intramuscularinjection, other pharmaceutically acceptable forms include, e.g. tabletsor other solids for oral administration; liposomal formulations; timerelease capsules; and any other form currently used.

A further object of the invention relates to a method for screening adrug suitable for the treatment of beta-thalassemia comprising the stepsconsisting of i) providing a candidate compound, ii) testing whether thecandidate compound for its ability to inhibit XPO1 activity orexpression and iii) selecting the candidate compound that is able toinhibit XPO1 activity or expression.

The invention will be further illustrated by the following figures andexamples. However, these examples and figures should not be interpretedin any way as limiting the scope of the present invention.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1: Expression of XPO1 gene in CD36+ erythroid precursors(immunoblot) N=Nucleus, C=Cytoplasm. From day 3 to day 6 of CD36+culture.

FIG. 2: Expression of XPO1 gene (and HSP70) in CD36+ erythroidprecursors (fluorescence microscopy).

FIG. 3A. KPT is not toxic for CD36+ erythroid progenitors from cordblood at day 6 (D6) of culture, when diluted at concentrations 33 nM,100 nM, 333 nM and 1000 nM in CD36+ culture media, with normalconcentrations of erythropoietin (EPO), until 20 h of treatment.

FIG. 3B. EPO starvation during 20 h results in 80% of cell death.Treatment of CD36+D6 with KPT at 100 nM, 333 nM during EPO starvationdecreases cell death, of 49% and 39% respectively, compared to theabsence of KPT treatment.

FIG. 4: KPT induces HSP70 nuclear retention in CD36+D6 from cord blood,in conditions of EPO starvation, after 20 h of treatment.

FIG. 5: In beta-thalassemia CD36+D8 cells, KPT induces HSP70 nuclearlocalization after 72 h of treatment at 100 nM compared to non treatedcells.

FIG. 6. CD36+ cells, derived from circulating CD34+ cells from Betathalassemia major patient, were treated or not with KPT molecule (KPT0=DMSO, KPT 100=100 nM, KPT 1000=1000 nM), and analysed at 24 h, 48 h,72 h and 96 h of treatment. Day 0 of treatment corresponds to day 5 ofCD36+ culture. a) Proliferation of CD36+ cells between 24 h and 96 h,for the different conditions of KPT treatment. b) Cell death of CD36+cells between 48 h and 96 h, for the different conditions of KPTtreatment. c) Flow cytometry analysis of Mean fluorescence intensity(MFI) for Band 3 protein, in CD36+ cells, in the different conditions ofKPT treatment. d) MGG count of immature, polychromatophiles andacidophiles+reticulocytes at 96 h of treatment, for the differentconditions of KPT treatment and e) corresponding maturation indexes.

FIG. 7: CD36+ cells, derived from circulating CD34+ cells from Betathalassemia major patient, were treated or not with KPT molecule (KPT0=DMSO, KPT 100=100 nM, KPT 1000=1000 nM), and analysed at 24 h, 48 h,and 72 h of treatment. Day 0 of treatment corresponds to day 6 of CD36+culture. a) Proliferation of CD36+ cells between 24 h and 72 h, for thedifferent conditions of KPT treatment. b) Cell death of CD36+ cellsbetween 24 h and 72 h, for the different conditions of KPT treatment. c)Flow cytometry analysis of MFI for Band 3 protein, in CD36+ cells, inthe different conditions of KPT treatment. d) Flow cytometry analysis ofthe number of small, alive, CD36+ cells positive for fœtal haemoglobinin the different conditions of KPT treatment. e) MGG count of immature,polychromatophiles and acidophiles+reticulocytes at 48 h of treatment,for the different conditions KPT 0 and KPT 100 nM and f) correspondingmaturation indexes.

FIG. 8. CD36+ cells from the same experiment as FIG. 8, at 24 h oftreatment. Percentages of cells containing HSP70 located in the nucleus(black) or in the cytoplasm (grey), gated on the total population ofcells analyzed, for the different treatment conditions: KPT 0 (a), KPT100 nM (b) and KPT 1000 nM (c). d) HSP70 cytoplasm/nuclear ratio for thethree treatment conditions e) Mean intensity and standard deviation forGATA1 transcription factor fluorescence.

EXAMPLE 1

Background

It has been published by our lab (Ribeil et al., Nature 2007) thatLeptomycin B (LMB), a chemical inhibitor of XPO1, induces HSP70 nuclearretention in erythroid precursor cells. This observation suggests thatXPO1 might be involved in the nuclear export of HSP70 protein inerythroid precursor.

During erythroid precursor cell differentiation, the nuclearaccumulation of HSP70 protein is essential. In fact, in the nucleus,HSP70 protects GATA1 transcription factor from Caspase-3 degradation.

In the case of two pathologies with dyserythropoiesis: myelodysplasticsyndrome (MDS) and beta-thalassemia, a defect in nuclear localization ofHSP70 in erythroid progenitor cells has been assessed (Frisan et al.Blood 2012, Arlet et al unpublished data). Thus, we suggest that itwould be of interest to repress XPO1-induced HSP70 nuclear export torestore HSP70 protein nuclear localization in erythroid precursorscells, and therefore improve erythroid precursors differentiation.

KPT-330 (Selinexor*) is the first selective inhibitor of XPO1 andproduced by Karyopharm. In vitro analysis and safety studies in humanpatients have shown a good tolerability of the molecule.

We suggest assessing the effect of KPT molecule (KPT-251 suitable for invitro tests) both in beta-thalassemia and MDS erythroid precursor cells.

Material and Methods:

Material

Umbilical cord blood units from normal full-term deliveries wereobtained, after informed mother's consent, from the Obstetrics Unit ofHôpital Necker-Enfants Malades. CD36+ erythroid progenitors, generatedfrom 7 days IL-6+IL3+SCF− cultured CD34 progenitors isolated from cordblood (Miltenyi CD34 Progenitor Cell Isolation Kit), were cultured inthe presence of IL3+SCF+Epo in IMDM (Gibco cell culture) supplementedwith 15% BIT 9500 (Stem Cell Technologies).

Immunoblot Analyses

Separate cytoplasmic and nuclear protein fractions were extracted fromerythroid progenitors using NE-PER Nuclear and cytoplasmic extractionreagents (Thermo Scientific), following manufacturer's protocol.

40 μg of proteins of nuclear or cytoplasmic extracts were resolved on14% acrylamide gels and analysed by immunoblotting. Antigens werevisualized by chemiluminescence using SuperSignal West Dura (ThermoScientific).

Reagents

Antibodies used are anti HDAC mouse clone 3F3 #05-814 (Millipore), antiHSP70 rabbit ADI SPA 812 (Enzo lifesciences), and anti CRM1 (XPO1)rabbit #ST1100 (Calbiochem).

Cell Permeabilisation and Labelling for Fluorescence Microscopy

5·10⁴ cells were spin on slides, acetone fixated, hydrated with cold1×PBS 1% BSA for 30 minutes, treated with formaldehyde for 15 min(Sigma), then with methanol (Prolabo) for 10 minutes at roomtemperature. Cells were then permeabilized with 1×PBS 0.2% triton X100(Sigma) for 10 min at 4° C., washed and incubated in 10% BSA for 30minutes. They were then sequentially incubated with antibodies dilutedin 1×PBS 1% BSA 0.1% tween (Sigma). Nuclei were stained with DAPI andslides were examined with a confocal laser microscope (LSM 700 CarlZeiss).

Results:

We investigated the expression of XPO1 protein in CD36+ erythroidprogenitors from cord blood by immunoblot (FIG. 1) and fluorescencemicroscopy (FIG. 2). In FIG. 1, XPO1 protein is present majority in thecytoplasm (C) and to a lesser extend in the nucleus (N) compartment ofCD36+ erythroid progenitors, from day 3 (D3) to day 6 (D6) of culture.In FIG. 2, the expression of XPO1 protein observed by immunoblotting isconfirmed by confocal microscopy, on permeabilized CD36+ erythroidprogenitors, at day 6 of culture in Epo. Here we report for the firsttime the expression of XPO1, a nuclear export protein of the betaimportin family, in CD36+ erythroid progenitor cells.

EXAMPLE 2

Data from FIG. 6 and FIG. 7 are from two different experiments on CD36+cells from a beta-thalassemia patient. Data from FIG. 8 have beenperformed with cells from FIG. 7 experiment. Beta-thalassemiaerythroblasts were generated in vitro from peripheral blood circulatingCD34+ cells from an adult patient with beta-thalassemia major (β⁰-TM).This study was performed according to the Helsinky Declaration withapproval from the ethics committee of our institution.

Treatment with KPT at 1000 nM shows decreased proliferation compared toKPT 0 and KPT 100 nM, only in the experiment where patient cells have ahigh proliferation rate in control condition (FIG. 6a and FIG. 7a ).Treatment with KPT at 1000 nM shows little cytotoxicity compared to KPT0 and KPT 100 nM (FIG. 6b , FIG. 7b ).

In vitro, maturation is arrested at the polychromatophilic stage inbeta-thalassemia erythroid progenitors. To quantify the maturationarrest in beta-thalassemia cells, we use an index of terminal maturationas the number (acidophilic cells+reticulocytes per slide)*100 divided bythe number of polychromatophilic cells per slide (Arlet J b et al.Nature oct 2014). We show that the maturation index of beta-thalassemiacells increases with KPT treatment at 100 nM and 1000 nM compared tocontrol condition KPT 0 (FIG. 6d, 6e , FIGS. 7e and 7f ).

Flow cytometry analyses show that the presence of KPT 100 nM or 1000 nMin culture media of CD36+ beta-thalassemia cells increases the meanfluorescence intensity (MFI) of Band 3 protein labelling, a marker ofterminal erythroid differentiation (Hu J et al. Blood April 2013), at 96h of treatment (FIG. 6c ), and at 72 h of treatment (FIG. 7c ).

In beta-thalassemia patients, the lack of beta chain synthesis iscompensated by an increased proportion of cells producing fetalhaemoglobin (HbF: composed of two alpha and to gamma subunits) and theonly surviving mature erythroblasts are HbF cells. Here we observe thatafter 72 hours of treatment with 1000 nM of KPT, the proportion of HbFpositive, among the population of small alive cells, is higher comparedto KPT 0 and KPT 100 nM conditions.

Amnis Technology enables to analyse fluorescence localization in a largenumber of cells beforehand labelled. Here we show the percentage ofcells positive for HSP70 labelling in the nucleus, or in the cytoplasm,for conditions KPT 0, 100 nM and 1000 nM (FIG. 8). We can observe thatthe fraction of cells positive for HSP70 in the nucleus increases in KPT100 nM and 1000 nM conditions (FIGS. 8b and 8c ), compared to KPT 0(FIG. 8a ).

This observation is also evidenced by the decrease in HSP70 cyto/nuclearfraction in conditions KPT 100 nM and 1000 nM compared to KPT 0 (FIG. 8d).

During terminal erythroid differentiation, HSP70 nuclear translocationis required to protect GATA1 transcription factor from the cleavage byactivated caspase 3. In beta-thalassemia erythroid progenitors, HSP70 issequestered in the cytoplasm by the excess of alpha globin chains (ArletJ b et al. Nature oct 2014). Thus, GATA1 is cleaved and this results inmaturation arrest and apoptosis of erythroid progenitors. Here we showthat the increasing fraction of cells with nuclear HSP70 coincides withan increase in the mean of fluorescence intensity of GATA1 labelling.

Taken together, these data show that treatment of CD36+ cells frombeta-thalassemia patients in vitro with KPT, a specific inhibitor ofXPO1 exportin, shows few cytotoxicity, and can improve terminalerythroid differentiation by repressing HSP70 export from the nucleus oferythroid progenitors. This nuclear translocation of HSP70 is associatedwith the protection of GATA1 erythroid transcription factor fromcleavage.

REFERENCES

Throughout this application, various references describe the state ofthe art to which this invention pertains. The disclosures of thesereferences are hereby incorporated by reference into the presentdisclosure.

The invention claimed is:
 1. A method of restoring or increasingerythrocyte maturation in a subject suffering from β-thalassemia major(β-TM) by preventing proteolytic inactivation of GATA-1, whereinpreventing is achieved by administering to the subject a compound thatis an XPO1 inhibitor of an XPO1 nuclear transporter, wherein the XPO1inhibitor is selected from the group consisting of:


2. The method of claim 1, wherein the XPO1 inhibitor is KPT-330 whichhas a formula:


3. A method of restoring or increasing erythrocyte maturation in asubject suffering from β-thalassemia major (β-TM) by preventingproteolytic inactivation of GATA-1, wherein preventing is achieved byadministering to the subject a compound that is an XPO1 inhibitor of anXPO1 nuclear transporter, wherein the XPO1 inhibitor has a formula


4. A method of treating beta-thalassemia in a subject in need thereof,or for promoting or restoring erythrocyte maturation and/or increasingthe production of (α/γ)₂ Hb erythrocytes in a subject suffering frombeta-thalassemia, comprising administering to the subject atherapeutically effective amount of an XPO1 inhibitor wherein the XPO1is selected from the group consisting of


5. A method of restoring or increasing erythrocyte maturation in asubject suffering from β-thalassemia major (β-TM) by preventingproteolytic inactivation of GATA-1, wherein preventing is achieved byadministering to the subject a compound that is an XPO1 inhibitor of anXPO1 nuclear transporter, wherein the XPO1 inhibitor is KPT-330 whichhas a formula:


6. A method of treating beta-thalassemia in a subject in need thereof,or for promoting or restoring erythrocyte maturation and/or increasingthe production of (α/γ)₂ Hb erythrocytes in a subject suffering frombeta-thalassemia, comprising administering to the subject atherapeutically effective amount of an XPO1 inhibitor wherein the XPO1inhibitor has a formula: